3.3 Screening of conjugates in cell proliferation activity and scratch wound healing assays
The efficacy of FGF2 conjugates for wound healing can be assessed by measuring the cell proliferation, migration, and vasculogenesis of dermal fibroblasts and endothelial cells (Rabuka, 2010; van Horssenet al. , 2006). Here, human dermal fibroblast cells (HDFs) and human umbilical vein endothelial cells (HUVECs) were used as in vitro models to screen and compare the bioactivities of these conjugates.
First, the dose-dependent effects of PEG-FGF2 conjugates on cell proliferation of HUVECs were investigated by CCK-8 assay. The cells were incubated with various treatment conditions at concentrations of 0.1, 1, and 10 ng·mL-1 of protein for 24 h. Compound 6 promoted significantly greater cell proliferation than all the other compounds, including native FGF2, even at a low concentration (0.1 ng·mL-1), with the highest pro-proliferation activity at a concentration of 10 ng·mL-1 (Figure 3A ). We also evaluated the cell proliferation activity of these conjugates by immunofluorescence staining for Ki67-positive HUVECs at 10 ng·mL-1. Compounds 5 and 6 showed a significant increase in the number of Ki67-positive cells compared with the PBS control, with a bigger effect for compound 6 than that for FGF2 (Figure 3 B-C ).
Next, a scratch wound healing assay was performed to evaluate the effects of PEG-FGF2 conjugates on cell migration of HUVECs. A scratch was made through a confluent layer of growth factor starved cells followed by the application of different compounds. Compared with FGF2 treatment, a significant reduction in wound area was observed upon treatment with compound 6 at 24 h, with migration of HUVECs increased by ~163% compared to only ~108% for FGF2 (Figure 3 D-E ). The activity of compound 5 is similar to that of FGF2. Overall, these results indicate that compound 6 can notably promote proliferation and migration of HUVECs relative to unmodified FGF2.
The same compounds were also tested using human dermal fibroblasts (HDFs). There was increased proliferation and migration of HDFs after treatment with compound 6 relative to treatment with other protein conjugates (Figure 4 A-C ). These results were consistent with the results for HUVECs, which indicated improved bioactivity of compound 6 to promote cell proliferation and scratch wound healing in both dermal fibroblasts and endothelial cells, which may be attributed to the long-acting effect of PEGylation.